Clinical diagnostics provide an essential aid to the physician for the diagnosis and monitoring of numerous pathologies and infectious diseases. Rapid and accurate identification of causative agents of a myriad of different human pathophysiological conditions is a paramount requisite to effective treatment.
A biological sample generally is taken from the patient, most often at the request of a physician, and sent to a medical laboratory for analysis to establish or confirm a diagnosis of clinical symptoms. A physician may suspect a particular causative agent upon physical examination. However, certain symptoms may be characteristic of a plethora of different causative agents. Therefore, due to misdiagnoses of causative agents, patients may be treated non-efficaciously. Moreover, in other instances, a physician may request a certain diagnostic test to be performed on a clinical specimen wherein the test subsequently produces a negative result. Then, of course, further clinical samples and diagnostic testing is required. In many instances, due to the lack of timely and accurate diagnoses, patients' original conditions progress to the further detriment of treatability and to the well-being of the patient. Accurate clinical diagnosis is critical to specifically identify causative agents, and in many cases specific species of infectious agents which mediate pathophysiological conditions in a timely manner. Accordingly, a need indeed exists for methods for detecting and identifying distinct species, in a single container, from a genus of infectious agents.
Direct detection techniques for Herpes Simplex Virus (HSV), for example, can include viral culture, Tzanck's smear, antigen detection, direct fluorescent antibody analysis, and viral DNA detection. Cusini et al., 2001, J. Antimicrob. Chemother. 47 Suppl T1, 9-16.
The use of conventional PCR techniques with probe-based hybridization for the direct detection of HSV DNA have been reported for subtyping of clinical specimens. Cone et al., 1991, J. Infect. Dis. 164:757-60. Conventional PCR has significant advantages over viral culture procedures, as the virus does not have to be infective and HSV DNA can be detected in the late stages of genital lesions more efficiently. Lucotte et al., 1995, Mol. Cell Probes 9:287-90. Stocher et al. reported on the parallel detection of CMV, EBV, HSV-1, HSV-2, and VZV from cerebrospinal fluid, serum, and plasma samples utilizing the LightCycler real-time PCR technology. Stocher et al., 2003, J. Clin. Virol. 26:85-93. Two reports developed TaqMan assays for the parallel detection of members of the Herpesviridae family in multiple, separate assays and yet another two have reported on SYBR green real-time PCR assays for the parallel detection of HSV-1, HSV-2, or both in clinical specimens. van Doornum et al., 2003, J. Clin. Microbiol. 41:576-80; Weidmann et al., 2003, J. Clin. Microbiol. 41:1565-8; Nicoll et al., 2001, J. Virol. Methods 96:25-31; Schmutzhard et al., 2004, J. Clin. Virol. 29:120-6. A recent study, for example, was published which analyzed the presence of HSV in more than 36,000 mucosal secretions found the detection rate of HSV DNA by real-time PCR techniques to be 12.1% of the samples versus 3.0% detected by viral culture. The method employed, however, could not discriminate between HSV-1 and HSV-2. Wald et al., 2003, J. Infect Dis. 188:1345-51.
Particularly, the need exists in the industry for rapid and accurate methods for ascertaining whether a sample includes a first nucleic acid-containing entity or a second nucleic acid-containing entity, the ability, for example, to identify distinct species of nucleic acids corresponding to distinct species of infectious agents in a single container.